β-Lapachone Exerts Anticancer Effects by Downregulating p53, Lys-Acetylated Proteins, TrkA, p38 MAPK, SOD1, Caspase-2, CD44 and NPM in Oxaliplatin-Resistant HCT116 Colorectal Cancer Cells

β-lapachone (β-Lap), a topoisomerase inhibitor, is a naturally occurring ortho-naphthoquinone phytochemical and is involved in drug resistance mechanisms. Oxaliplatin (OxPt) is a commonly used chemotherapeutic drug for metastatic colorectal cancer, and OxPt-induced drug resistance remains to be solved to increase chances of successful therapy. To reveal the novel role of β-Lap associated with OxPt resistance, 5 μM OxPt-resistant HCT116 cells (HCT116-OxPt-R) were generated and characterized via hematoxylin staining, a CCK-8 assay and Western blot analysis. HCT116-OxPt-R cells were shown to have OxPt-specific resistance, increased aggresomes, upregulated p53 and downregulated caspase-9 and XIAP. Through signaling explorer antibody array, nucleophosmin (NPM), CD37, Nkx-2.5, SOD1, H2B, calreticulin, p38 MAPK, caspase-2, cadherin-9, MMP23B, ACOT2, Lys-acetylated proteins, COL3A1, TrkA, MPS-1, CD44, ITGA5, claudin-3, parkin and ACTG2 were identified as OxPt-R-related proteins due to a more than two-fold alteration in protein status. Gene ontology analysis suggested that TrkA, Nkx-2.5 and SOD1 were related to certain aggresomes produced in HCT116-OxPt-R cells. Moreover, β-Lap exerted more cytotoxicity and morphological changes in HCT116-OxPt-R cells than in HCT116 cells through the downregulation of p53, Lys-acetylated proteins, TrkA, p38 MAPK, SOD1, caspase-2, CD44 and NPM. Our results indicate that β-Lap could be used as an alternative drug to overcome the upregulated p53-containing OxPt-R caused by various OxPt-containing chemotherapies.


Introduction
β-lapachone (β-Lap) is a naturally occurring ortho-naphthoquinone compound, originally isolated from the lapacho tree (Tabebuia avellanedae), and can be synthesized from

Generation of 5 µM OxPt-Resistant HCT116 Cells
To further elucidate OxPt-R properties in wild-type p53-containing colorectal cancer, we generated 5 µM OxPt-resistant HCT116 cells from HCT116 cells via long-term treatment with OxPt for several months. As shown in Figure 1a,b, OxPt-R cells were treated with a gradually increasing OxPt concentration from 0.2 µM to 1 µM such that the indicated passages would not to have significant morphological changes and cytotoxicity compared to parental HCT116 cells; the resulting 1 µM OxPt-R cells were stimulated with a high concentration of 6.3 µM OxPt for two passages to strongly induce OxPt properties; 6.3 µM OxPt-R cells were stabilized with a lower concentration of 2.5 µM OxPt for 10 passages; and 2.5 µM OxPt-R cells were treated with 5 µM OxPt for 16 passages. As a result, 5 µM OxPtresistant stable HCT116 cells (HCT116-OxPt-R) were generated, and HCT116-OxPt-R cells at passage 16 (P16) were stored in a liquid nitrogen tank. Parental HCT116 cells and HCT116-OxPt-R cells (P16) were used in this study, and the results were confirmed through repeated experiments. First, we investigated the morphological differences between HCT116 and HCT116-OxPt-R cells from P17 to P22 via phase-contrast microscopy to find a novel OxPt-R-related phenotype. Notably, HCT116-OxPt-R cells appeared to be more complex than HCT116 cells, probably due to certain accumulated macromolecules produced through long-term treatment with OxPt ( Figure 1c). This fact suggests that complex morphological changes in HCT116-OxPt-R cells may be associated with OxPt-R properties against OxPtinduced cytotoxicity.
caspase-2, CD44 and NPM. Thus, we suggest that β-Lap could be used as a natural chemotherapeutic agent with low cytotoxicity for the treatment of various OxPt-R cancers through the inhibition of multiple OxPt-R-related proteins, including upregulated p53.

Generation of 5 μM OxPt-Resistant HCT116 Cells
To further elucidate OxPt-R properties in wild-type p53-containing colorectal cancer, we generated 5 μM OxPt-resistant HCT116 cells from HCT116 cells via long-term treatment with OxPt for several months. As shown in Figure 1a,b, OxPt-R cells were treated with a gradually increasing OxPt concentration from 0.2 μM to 1 μM such that the indicated passages would not to have significant morphological changes and cytotoxicity compared to parental HCT116 cells; the resulting 1 μM OxPt-R cells were stimulated with a high concentration of 6.3 μM OxPt for two passages to strongly induce OxPt properties; 6.3 μM OxPt-R cells were stabilized with a lower concentration of 2.5 μM OxPt for 10 passages; and 2.5 μM OxPt-R cells were treated with 5 μM OxPt for 16 passages. As a result, 5 μM OxPt-resistant stable HCT116 cells (HCT116-OxPt-R) were generated, and HCT116-OxPt-R cells at passage 16 (P16) were stored in a liquid nitrogen tank. Parental HCT116 cells and HCT116-OxPt-R cells (P16) were used in this study, and the results were confirmed through repeated experiments. First, we investigated the morphological differences between HCT116 and HCT116-OxPt-R cells from P17 to P22 via phase-contrast microscopy to find a novel OxPt-R-related phenotype. Notably, HCT116-OxPt-R cells appeared to be more complex than HCT116 cells, probably due to certain accumulated macromolecules produced through long-term treatment with OxPt ( Figure 1c). This fact suggests that complex morphological changes in HCT116-OxPt-R cells may be associated with OxPt-R properties against OxPt-induced cytotoxicity.

Morphological Phenotype
To elucidate the novel OxPt-R-related morphological phenotype in HCT116-OxP cells, we examined morphological changes between HCT116 and HCT116-OxPt-R ce via phase-contrast microscopy after hematoxylin staining. As shown in Figure 2, the ov all cell morphology was somewhat different, and the nuclear staining with hematoxy solution was stronger in HCT116-OxPt-R cells compared to HCT116 cells (compare pan a,b). Moreover, HCT116-OxPt-R cells contained abnormal nuclear membrane structu and more aggresomes or granules compared to HCT116 cells, especially in the areas in cated by arrows (compare panels a',b'; a'',b''; a''',b'''). This fact suggests that certain gresomes or granules produced in HCT116-OxPt-R cells may be a novel OxPt-R-rela phenotype that may be associated with the OxPt-R property.  (c) Morphological changes between HCT116 and HCT116-OxPt-R cells. HCT116 and HCT116-OxPt-R cells from passage 17 (P17) to P22 were analyzed via phase-contrast microscopy.

Morphological Phenotype
To elucidate the novel OxPt-R-related morphological phenotype in HCT116-OxPt-R cells, we examined morphological changes between HCT116 and HCT116-OxPt-R cells via phase-contrast microscopy after hematoxylin staining. As shown in Figure 2, the overall cell morphology was somewhat different, and the nuclear staining with hematoxylin solution was stronger in HCT116-OxPt-R cells compared to HCT116 cells (compare panels a,b). Moreover, HCT116-OxPt-R cells contained abnormal nuclear membrane structures and more aggresomes or granules compared to HCT116 cells, especially in the areas indicated by arrows (compare panels a , b ; a , b ; a , b ). This fact suggests that certain aggresomes or granules produced in HCT116-OxPt-R cells may be a novel OxPt-R-related phenotype that may be associated with the OxPt-R property.

Morphological Phenotype
To elucidate the novel OxPt-R-related morphological phenotype in HCT116-OxPt-R cells, we examined morphological changes between HCT116 and HCT116-OxPt-R cells via phase-contrast microscopy after hematoxylin staining. As shown in Figure 2, the overall cell morphology was somewhat different, and the nuclear staining with hematoxylin solution was stronger in HCT116-OxPt-R cells compared to HCT116 cells (compare panels a,b). Moreover, HCT116-OxPt-R cells contained abnormal nuclear membrane structures and more aggresomes or granules compared to HCT116 cells, especially in the areas indicated by arrows (compare panels a',b'; a'',b''; a''',b'''). This fact suggests that certain aggresomes or granules produced in HCT116-OxPt-R cells may be a novel OxPt-R-related phenotype that may be associated with the OxPt-R property.

Molecular Mechanisms
To elucidate the molecular mechanisms involved in OxPt-R, we examined apoptosisrelated protein levels via Western blot analysis in HCT116 and HCT116-OxPt-R cells treated with OxPt for 36 h. According to the result, HCT116-OxPt-R cells contained upregulated p53, Noxa and γ-H2AX and downregulated caspase-9 and XIAP compared to HCT116 cells (Figure 4a,b; compare lanes 1,5). The upregulation of p53 and Noxa and the downregulation of capase-9 and XIAP by 5 μM OxPt were significant in HCT116 cells but not in HCT116-OxPt-R cells, indicating relevance to drug resistance against 5 μM OxPt in HCT116-OxPt-R cells (Figure 4a,b; compare lanes 1,2 and 5,6). However, p53 and Noxa Error bars represent standard deviation of the mean. Statistical significance between non-treated (NT) control and drug-treated sample was determined using Student's t-test, * p < 0.05 (c,d).

Molecular Mechanisms
To elucidate the molecular mechanisms involved in OxPt-R, we examined apoptosisrelated protein levels via Western blot analysis in HCT116 and HCT116-OxPt-R cells treated with OxPt for 36 h. According to the result, HCT116-OxPt-R cells contained upregulated p53, Noxa and γ-H2AX and downregulated caspase-9 and XIAP compared to HCT116 cells (Figure 4a,b; compare lanes 1, 5). The upregulation of p53 and Noxa and the downregulation of capase-9 and XIAP by 5 µM OxPt were significant in HCT116 cells but not in HCT116-OxPt-R cells, indicating relevance to drug resistance against 5 µM OxPt in HCT116-OxPt-R cells (Figure 4a,b; compare lanes 1, 2 and 5, 6). However, p53 and Noxa were also upregulated by 20 µM OxPt treatment in HCT116-OxPt-R cells, but not as much as in HCT116 cells, indicating partial activation of apoptotic signaling with 20 µM OxPt in HCT116-OxPt-R cells (Figure 4a; compare lanes 4,8). Notably, γ-H2AX was upregulated by OxPt in a concentration-dependent manner in HCT116 cells, whereas upregulated γ-H2AX in HCT116-OxPt-R cells was rather downregulated by OxPt in a concentrationdependent manner (Figure 4a, third panel). Moreover, Akt, caspase-9 and XIAP were significantly downregulated by OxPt in HCT116 cells, but these proteins were not affected by 5 µM OxPt, and were somewhat upregulated by 20 µM OxPt in HCT116-OxPt-R cells ( Figure 4b). Collectively, these results suggest that OxPt-R-related molecular mechanisms in HCT116-OxPt-R cells are related to the inhibition of p53-dependent apoptotic signaling. were also upregulated by 20 μM OxPt treatment in HCT116-OxPt-R cells, but not as much as in HCT116 cells, indicating partial activation of apoptotic signaling with 20 μM OxPt in HCT116-OxPt-R cells (Figure 4a; compare lanes 4,8). Notably, γ-H2AX was upregulated by OxPt in a concentration-dependent manner in HCT116 cells, whereas upregulated γ-H2AX in HCT116-OxPt-R cells was rather downregulated by OxPt in a concentration-dependent manner (Figure 4a, third panel). Moreover, Akt, caspase-9 and XIAP were significantly downregulated by OxPt in HCT116 cells, but these proteins were not affected by 5 μM OxPt, and were somewhat upregulated by 20 μM OxPt in HCT116-OxPt-R cells (Figure 4b). Collectively, these results suggest that OxPt-R-related molecular mechanisms in HCT116-OxPt-R cells are related to the inhibition of p53-dependent apoptotic signaling.
Notably, most of the OxPt-R-related proteins identified using the antibody array were upregulated in HCT116-OxPt-R cells, as shown in Figure 5, which may be related to the accumulation of certain aggresomes or granules.

Verification of OxPt-R-Related Proteins Identified via Antibody Array
To confirm the results in Table 1, the protein samples isolated for antibody array were analyzed via Western blot using the indicated antibodies. As expected, the protein levels of Lys-acetylated proteins, TrkA, p38 MAPK, SOD1, caspase-2, CD44 and NPM were upregulated in HCT116-OxPt-R cells comparted to HCT116 cells ( Figure 6).

Verification of OxPt-R-Related Proteins Identified via Antibody Array
To confirm the results in Table 1, the protein samples isolated for antibody array analyzed via Western blot using the indicated antibodies. As expected, the protein of Lys-acetylated proteins, TrkA, p38 MAPK, SOD1, caspase-2, CD44 and NPM we regulated in HCT116-OxPt-R cells comparted to HCT116 cells ( Figure 6). Figure 6. Verification of OxPt-R-related proteins identified via antibody array: HCT11 HCT116-OxPt-R cells were grown for 72 h, and protein samples for antibody array were pre as described in "Materials and Methods", and then, analyzed via Western blot using the ind antibodies.

Gene Ontology Analysis
The results in Table 1 were also confirmed via Western blot analysis on who extracts of HCT116 and HCT116-OxPt-R prepared using 1× SDS sample buffer. A pected, Lys-acetylated proteins, TrkA, p38 MAPK, SOD1 and caspase-2 were upregu in the whole-cell extract of HCT116-OxPt-R compared to HCT116, whereas CD4 NPM were somewhat downregulated in the whole-cell extract of HCT116-OxPt-R v unknown mechanism. Thus, we analyzed the gene ontology for the newly iden OxPt-R-related proteins using the DAVID database, except for CD44 and NPM. As s in Figure 7, the OxPt-R properties of HCT116-OxPt-R cells were significantly asso with: the positive regulation of gene expression through the upregulation of MA (p38-β MAPK), calreticulin and Nkx-2.5; the macromolecular complex through the u ulation of NTRK1 (TrkA), Nkx-2.5 and SOD1; and chaperone binding through the u ulation of calreticulin and SOD1. These results suggest that the morphological phen of HCT116-OxPt-R cells may be more complex due to the upregulation of OxPt-R-r proteins associated with the positive regulation of gene expression, the macromole complex and chaperone binding. Figure 6. Verification of OxPt-R-related proteins identified via antibody array: HCT116 and HCT116-OxPt-R cells were grown for 72 h, and protein samples for antibody array were prepared as described in "Materials and Methods", and then, analyzed via Western blot using the indicated antibodies.

Gene Ontology Analysis
The results in Table 1 were also confirmed via Western blot analysis on whole-cell extracts of HCT116 and HCT116-OxPt-R prepared using 1× SDS sample buffer. As expected, Lys-acetylated proteins, TrkA, p38 MAPK, SOD1 and caspase-2 were upregulated in the whole-cell extract of HCT116-OxPt-R compared to HCT116, whereas CD44 and NPM were somewhat downregulated in the whole-cell extract of HCT116-OxPt-R via an unknown mechanism. Thus, we analyzed the gene ontology for the newly identified OxPt-R-related proteins using the DAVID database, except for CD44 and NPM. As shown in Figure 7, the OxPt-R properties of HCT116-OxPt-R cells were significantly associated with: the positive regulation of gene expression through the upregulation of MAPK11 (p38-β MAPK), calreticulin and Nkx-2.5; the macromolecular complex through the upregulation of NTRK1 (TrkA), Nkx-2.5 and SOD1; and chaperone binding through the upregulation of calreticulin and SOD1. These results suggest that the morphological phenotype of HCT116-OxPt-R cells may be more complex due to the upregulation of OxPt-R-related proteins associated with the positive regulation of gene expression, the macromolecular complex and chaperone binding.

Anticancer Effect by β-Lap in HCT116-OxPt-R Cells
To elucidate the anticancer effect of β-Lap in relation to OxPt-R, we investigated the effect of β-Lap on cell viability and morphological changes in HCT116 and HCT116-OxPt-R cells via CCK-8 assay and phase-contrast microscopy. As a result, the anticancer effect induced by β-Lap was significantly higher in HCT116-OxPt-R cells than in HCT116 cells, which caused more cytotoxicity and morphological changes in HCT116-OxPt-R cells (Figure 9a-c). Higher morphological changes by β-Lap in HCT116-OxPt-R cells than in HCT116 cells were demonstrated via phase-contrast microscopy after hematoxylin staining (Figure 9d).

Anticancer Effect by β-Lap in HCT116-OxPt-R Cells
To elucidate the anticancer effect of β-Lap in relation to OxPt-R, we investigated the effect of β-Lap on cell viability and morphological changes in HCT116 and HCT116-OxPt-R cells via CCK-8 assay and phase-contrast microscopy. As a result, the anticancer effect induced by β-Lap was significantly higher in HCT116-OxPt-R cells than in HCT116 cells, which caused more cytotoxicity and morphological changes in HCT116-OxPt-R cells (Figure 9a-c). Higher morphological changes by β-Lap in HCT116-OxPt-R cells than in HCT116 cells were demonstrated via phase-contrast microscopy after hematoxylin staining (Figure 9d).

Discussion
Oxaliplatin (OxPt) is a widely used chemotherapeutic agent in the treatment of various cancers, including metastatic colorectal cancer. However, OxPt-induced drug resistance (OxPt-R) still remains a major issue to be solved to increase chances of successful cancer therapy. β-lapachone (β-Lap), a natural phytochemical, is expected to be developed as a chemotherapeutic agent for drug resistance caused by long-term treatment due to its potent anticancer activities [24,27,28]. In this study, we investigated the novel role of β-Lap related to oxaliplatin resistance (OxPt-R) in wild-type p53-containing HCT116 colorectal cancer cells. As a result, the anticancer activity by β-Lap was significantly higher in HCT116-OxPt-R cells resistant to 5 µM OxPt than in HCT116 cells; the anticancer mechanism of β-Lap in HCT116-OxPt-R cells was associated with the downregulation of OxPt-R-related proteins, including p53, Lys-acetylated proteins, TrkA, p38 MAPK, SOD1, caspase-2, CD44 and NPM (Figures 9 and 10).
Many studies that elucidate OxPt-R-related mechanisms have been accomplished in various OxPt-R cancer cells, which are generated via long-term treatment with OxPt, with somewhat different experimental procedures depending on the cell type and researcher [17,[29][30][31][32][33][34]. However, further studies on OxPt-R-related morphological properties are still essentially needed to better understand OxPt-R mechanisms. In the present study, we generated HCT116-OxPt-R cells resistant to 5 µM OxPt using our own method, as described in Figure 1b, and found that the intracellular structure of HCT116-OxPt-R is more complex than that of HCT116 cells, at least in part, due to OxPt-R-induced aggresomes or granules (Figures 1c and 2). To better understand OxPt-R-related molecular mechanisms, we identified new OxPt-R-related proteins by analyzing upregulated or downregulated proteins in HCT116-OxPt-R cells compared to HCT116 cells through signaling explorer antibody array analysis. As shown in Table 1 and Figure 5, NPM1, CD37, NKX2-5, SOD1, H2BS1, CALR, MAPK11, CASP2, CDH9, MMP23B, ACOT2, COL3A1, NTRK1, RPS27, CD44 and ITGA5 were upregulated more than two-fold in HCT116-OxPt-R cells compared to HCT116 cells, whereas CLDN3, PRKN and ACTG2 were downregulated more than two-fold. In addition, gene ontology analysis of the newly identified OxPt-R-related proteins suggested that OxPt-R properties in HCT116-OxPt-R cells may be related to the positive regulation of gene expression, the macromolecular complex and chaperone binding (Figure 7). These results indicate that the upregulated OxPt-R-related proteins may be implicated in the intracellular complexity of HCT116-OxPt-R compared to HCT116 cells and the production of OxPt-R-related aggresomes in HCT116-OxPt-R cells, supporting the results of Figures 1c and 2.
The tumor suppressor p53 plays an important role in DNA repair, cell cycle arrest, cell death, senescence, differentiation and metabolism in a transcriptional activity-dependent or -independent manner in response to cellular stress [35]. Colorectal cancer is one of the most common malignancies with high prevalence, and is associated with p53 mutations and impaired wild-type p53 function [36,37]. It has been shown that OxPt-induced anticancer activity was significantly higher in wild-type p53-containing colorectal cancer cells than in mutant p53 or null-type p53, whereas OxPt-R properties were higher in null-type p53-containing colorectal cancer cells than wild-type p53 [38,39], suggesting that OxPtinduced anticancer activity and OxPt-R properties may be controlled by p53 status. In the present study, we show that OxPt-R properties in HCT116-OxPt-R cells were induced in the presence of upregulated p53-mediated apoptotic signaling, as well as upregulated survivinand ERK-mediated survival signaling (Figures 4a and 10a). Moreover, our results showed that OxPt-R properties in HCT116-OxPt-R cells could be controlled by new OxPt-R-related proteins, including Lys-acetylated proteins, TrkA, p38 MAPK, caspase-2, CD44 and NPM ( Figure 6). It has been shown that a single nucleotide deletion in the amino acid 382 of p53 occurred in OxPt-R cells generated from KB cells that are a subclone of human cervical carcinoma HeLa cells, and resulted in the large cytoplasmic accumulation of p53, leading to functional defects in p53 [40]. Collectively, our results suggest that OxPt-R properties in HCT116-OxPt-R cells may be related to the upregulation of functionally inactivated p53 due to probably certain mutations caused by long-term treatment with OxPt; moreover, OxPt-R properties may occur by overcoming partially activated apoptotic signaling in HCT116-OxPt-R cells through the upregulation of OxPt-R-related survival proteins.
In addition to camptothecin (CPT), β-Lap is known as a novel DNA topoisomerase I inhibitor; however, the action mode of β-Lap is different from that of CPT, and β-Lap inhibits topoisomerase I-mediated DNA cleavage induced by CPT [41,42]. β-Lap has been shown to induce apoptosis in human colon cancer cells, promyelocytic leukemia cells and prostate cancer cells, regardless of p53 status [4,5]; however, β-Lap-induced apoptosis was also associated with the activation of p53-dependent apoptotic signaling in human prostate epithelial cells through the phosphorylation of p53, the induction of Bax, and the activation of caspases [43]. In the present study, HCT116-OxPt-R cells appeared to be highly resistant to 5 µM OxPt, but not to topoisomerase I inhibitors CPT and β-Lap; cytotoxicity caused by CPT was similar between HCT116 and HCT116-OxPt-R cells, but cytotoxicity caused by β-Lap was significantly higher in HCT116-OxPt-R cells than in HCT116 cells (Figures 3  and 9). In addition, our results demonstrated that p53 was greatly upregulated by 5 µM OxPt, but not by β-Lap, in HCT116 cells; moreover, the upregulated p53 in HCT116-OxPt-R cells was downregulated by β-Lap in a concentration-dependent manner ( Figure 10). Since the anticancer activity of β-Lap is associated with the downregulation of mutant p53 [4,27], we strongly suggest that upregulated p53 in HCT116-OxPt-R cells may be mutated during long-term treatment with OxPt for several months, and is involved in OxPt-R properties in HCT116-OxPt-R cells.
It is known that lysine 9-acetylated histone H3 is upregulated at the MDR1 promoter in doxorubicin-resistant MCF-7 human breast cancer cells [44]; additionally, lysine 68-acetylated manganese superoxide dismutase (MnSOD) is associated with cisplatin and doxorubicin resistance due to aberrant mitochondria metabolism in MCF-7 cells [45]. In the present study, our results showed that lysine 15-acetylated histone H2B and unknown Lys-acetylated proteins were associated with OxPt-R properties in HCT116-OxPt-R cells (Table 1, Figure 6). Notably, the upregulation of Lys-acetylated proteins in HCT116-OxPt-R cells was significantly reduced by β-Lap in a concentration-dependent manner, but not in HCT116 cells, suggesting a novel anticancer mechanism of β-Lap associated with OxPt-R (Figure 10b). It is known that the dual-activation of TrkA and CD44 by NGF is involved in drug resistance to lestaurtinib in MDA-MB-231 human breast cancer cells [46]; the increase in phosphorylated p38 MAPK is associated with OxPt-R occurring in H29-D4 human colorectal cancer cells [47]; the increased caspase-2 expression in certain types of tumor has been linked to the promotion of tumorigenesis [48]; CD44 promotes resistance to etoposide-induced apoptosis in SW620 human colon cancer cells [49]; and the decreased NPM caused by trastuzumab reduces the drug resistance of gastric cancer to OxPt [50]. In the present study, we found that the downregulation of p53, survivin, ERK, Lys-acetylated proteins, TrkA, p38 MAPK, SOD1, caspase-2, CD44 and NPM by β-Lap was significantly higher in HCT116-OxPt-R cells than in HCT116 cells, and this phenomenon was associated with the higher anticancer activity of β-Lap in HCT116-OxPt-R than in HCT116 cells (Figures 9 and 10).
Taken together, we generated HCT116-OxPt-R cells that are highly resistant to 5 µM OxPt from wild-type p53-containing HCT116 cells, and found that OxPt-R properties are related to certain aggresomes produced during long-term treatment with OxPt. To better understand OxPt-R-related molecular mechanisms, we identified new OxPt-R-related proteins through signaling explorer antibody array analysis, and then, compared the anticancer effect and molecular mechanisms of β-Lap between HCT116 and HCT116-OxPt-R cells. We show here that β-Lap exerts more cytotoxicity and morphological changes in HCT116-OxPt-R cells than in HCT116 cells through the downregulation of OxPt-R-related proteins such as p53, Lys-acetylated proteins, TrkA, p38 MAPK, SOD1, caspase-2, CD44 and NPM. Therefore, we propose that β-Lap could be used as an effective chemotherapeutic agent for various OxPt-R-related cancer treatments, especially in the presence of upregulated p53 and other OxPt-R-related proteins during long-term chemotherapy.

Phase-Contrast Microscopy of Hematoxylin-Stained Cells
Hematoxylin (cationic) staining is used to detect nuclei (DNA, RNA and acid nucleoprotein) [51]. Cells grown on a 6-well plate were washed with PBS, fixed with 4% formaldehyde solution, washed with PBS, and then, stained with hematoxylin solution for 24 h at RT by gently shaking. The cells were washed with PBS and were analyzed in the presence of 90% glycerol/PBS solution via phase-contrast microscopy (EVOS XL Core, Thermo Fisher Scientific) at a 20× objective (Inf Plan Fluor 20× LWD, 0.45 NA/7.1 WD) with 300× amplification.

Cell Viability Analysis
Cells grown on a 24-well dish were incubated with maintenance medium containing 10% CCK-8 reagent for 1.5 h in a 37 • C CO 2 incubator. The reaction solution (100 µL each) was then transferred to a 96-well dish and was analyzed by measuring the absorbance at OD 450 nm using a microplate reader (SoftMax Pro 5 Software, Molecular Devices, San Jose, CA, USA).

Western Blot Analysis
Whole cells (attached and floating cells) were extracted with 1× SDS sample buffer and were boiled for 5 min at 95 • C. The resultant proteins were separated using SDS-PAGE and transferred to an NC membrane at 30 mA for 13-15 h. After washing with PBST (0.1% Tween-20, PBS) twice for 1 h, the membrane was blocked for 30 min at RT in blocking buffer (3% skim milk, 0.1% Tween-20, PBS), and then, incubated with primary antibody in blocking buffer at 4 • C overnight. The blot was then washed with PBST three times for 10 min, and incubated with an HRP-conjugated secondary antibody in blocking buffer for 2 h at RT. After being washed with PBST, the blot was analyzed using the ECL Western blot detection system.

Sample Preparation for Antibody Array
HCT116 and HCT116-OxPt-R cells were grown for 72 h and washed with PBS twice, and cell pellets were stored in a −70 • C freezer until use. To prepare protein samples for antibody array, cell pellets were extracted using protein extraction buffer (Fullmoon biosystems, Sunnyvale, CA, USA) containing 1% protease inhibitor cocktail (Sigma, St. Louis, MO, USA) and 1% phosphatase inhibitor cocktail (Sigma, St. Louis, MO, USA) and lysis beads (Fullmoon biosystems, Sunnyvale, CA, USA). After extraction, protein solution was purified using gel matrix column that was included in antibody array assay kit (Fullmoon biosystems, Sunnyvale, CA, USA). Concentration of purified sample was measured via BCA protein assay kit (Pierce, Rockford, IL, USA) using Multi-Skan FC (Thermo Fisher Scientific, Rockford, IL, USA). Additionally, purity of purified sample was confirmed on UV spectrum.

Signaling Explorer Antibody Array Analysis
Signaling explorer antibody array analysis result simultaneously shows the expression of 1358 proteins involved in 20 cell signaling pathways. To identify new OxPt-R-related proteins through signaling explorer antibody array analysis, 50 µg of protein sample prepared for antibody array was filled up to 75 µL with labeling buffer, and we treated 3 µL of the 10 µg/µL biotin/DMF solution at RT for 90 min with mixing. The sample was treated with 35 µL of stop reagent and incubated at RT for 30 min with mixing. The antibody microarray slide (Fullmoon biosystems, Sunnyvale, CA, USA) was treated with 30 mL of blocking solution in a Petri dish, incubated on a shaker at 60 rpm for 30 min at RT and washed with distilled water three times. The slide was rinsed with Milli-Q-grade water. The labeled sample was mixed in 6 mL of coupling solution. The blocked array slide was incubated with coupling mixture on shaker at 60 rpm for 2 h at RT in a coupling dish. The slide was washed 6 times with 30 mL of washing solution in a Petri dish on a shaker at 60 rpm for 5 min. Additionally, the slide was rinsed with Milli-Q-grade water. A total of 30 uL of 0.5 mg/mL Cy3-streptavidin (GE Healthcare, Chalfont St. Giles, UK) was mixed in 30 mL of detection buffer. The coupled array slide was treated with detection mixture in a Petri dish on a shaker at 60 rpm for 20 min at RT. The slide was washed 6 times with 30 mL of washing solution in a Petri dish on a shaker at 60 rpm for 5 min. Additionally, the slide was rinsed with Milli-Q-grade water. Slide scanning was performed using a GenePix 4100A scanner (Axon Instruments, Scottsdale, AZ, USA). The slides were completely dried before scanning and scanned within 24-48 h. The slides were scanned at a 10 µm resolution, optimal laser power and PMT. After obtaining the scan image, the scans were gridded and quantified with GenePix 7.0 Software (Axon Instruments, Scottsdale, AZ, USA). The data on protein information were annotated using UniProt DB.

Statistical Analysis
Results for cell viability are presented as mean ± standard deviation of the mean. Statistical significance between control and sample was determined using Student's t-test. Values of p < 0.05 are considered statistically significant.